CRACI-Seq for Quantitative RNA D Modification Profiling
Dihydrouridine (D) is the second most abundant modified nucleoside in tRNA, yet quantitative detection has remained inaccessible — until now. Existing methods (AlkAniline-Seq, D-seq, Rho-seq) rely on RT truncation that cannot measure modification stoichiometry or resolve densely modified tRNA D-loops.
CRACI-Seq (Chemical Reduction Assisted Cytosine Incorporation sequencing) changes this. KBH₄ reduction converts D sites into T→C misincorporation signatures readable at single-nucleotide resolution, achieving ~96% conversion across all sequence contexts with quantitative stoichiometry.
Single-base resolution — precisely maps D to individual nucleotide positions in the tRNA D-loop
Quantitative stoichiometry — absolute D modification fraction per site via motif-specific calibration
Full read-through of clustered D sites — internal misincorporation, not RT truncation
Mild chemistry preserves RNA integrity — KBH₄ reduction without backbone cleavage or base loss
tRNA + mRNA coverage — D profiling across both substrate types from one service platform
Dihydrouridine (D) is formed by reduction of the C5–C6 double bond of uridine, producing a saturated, non-aromatic ring that is structurally unique among RNA modifications. This saturation eliminates base-stacking ability and introduces local backbone flexibility critical for proper tRNA D-loop folding and tertiary structure.
D is installed by four dihydrouridine synthase (DUS) enzymes in mammals — DUS1L, DUS2L, DUS3L, and DUS4L — each recognizing specific positions within the tRNA D-loop. Recent research links D modification to cancer biology: DUS2L overexpression in NSCLC suppresses ferroptosis through tRNACys-dependent translational regulation; DUS1L expression correlates with glioblastoma prognosis; and DUS4L has been shown tumorigenic in lung cancer models. These findings have positioned DUS enzymes as emerging therapeutic targets and D modification itself as a pharmacodynamic biomarker of interest.
Our service delivers D modification maps across tRNAs and mRNAs, with per-nucleotide stoichiometry values, DUS substrate assignment support, and differential D analysis between experimental conditions.
Technology
Technical Principle: Base-Resolution Quantitative D Mapping
CRACI-Seq detects dihydrouridine through a three-step process that converts each D site into a measurable T→C misincorporation signal.
1) KBH₄ reduction — Total RNA is treated with potassium borohydride under neutral conditions, selectively reducing the C5–C6 double bond of D to tetrahydrouridine. The reaction completes within 3 hours without backbone cleavage or base loss, confirmed by LC-MS/MS. 2) HIV RT misincorporation — Reverse transcription under a skewed dGTP/dNTP ratio (1 mM/100 μM) drives preferential incorporation of C (rather than A) at tetrahydrouridine positions, producing a T→C mismatch in cDNA. The misincorporation rate averages ~96% across all 256 NNDNN motifs, with ≤4–15% background at undmodified U. 3) Calibration-based quantification — Motif-specific calibration curves convert raw T→C rates to absolute D stoichiometry, providing the fraction of RNA molecules modified at each position rather than a binary present/absent call. A molecular tag in the 3′ adapter enables PCR duplicate removal, ensuring mutation rates reflect the original RNA template population.
Services
Service Options: tRNA D Profiling and mRNA D Discovery
CRACI-Seq can be applied to two RNA substrate types depending on your research question.
Service Option
RNA Substrate
Typical D Sites Detected
Recommended Applications
tRNA D Profiling
Small RNA (<200 nt)
Positions 16, 17, 20, 20a, 20b, 47 across cytoplasmic tRNAs; mitochondrial tRNA D sites
DUS writer assignment; tRNA structure-function studies; cancer tRNA epitranscriptomics; cross-species D conservation analysis
mRNA D Discovery
PolyA+ RNA
Rare, low-stoichiometry D sites (10–40% range)
Exploratory studies of D as a new mRNA epitranscriptomic mark; DUS perturbation effects on mRNA modification
Both options include the same core CRACI chemistry, library construction, and sequencing workflow. The primary difference is the RNA input fraction: tRNA profiling enriches for small RNA to maximize D detection sensitivity in tRNAs (where D is most abundant), while mRNA D discovery uses polyA selection to investigate the recently discovered presence of D in messenger RNA. For studies requiring comprehensive coverage of both tRNA and mRNA D landscapes, we recommend requesting both service options from the same biological sample.
Our CRACI-Seq service follows a standardized workflow with QC checkpoints at each stage.
1. RNA QC and Sample Assessment — QC Checkpoint: RNA integrity verified by Bioanalyzer or Fragment Analyzer (RIN > 7 for total RNA); concentration quantified by fluorometry (Qubit); purity assessed by spectrophotometry (OD260/280 = 1.8–2.2). Sample type verified against service option requirements.
2. Size Selection / PolyA Enrichment — For tRNA profiling: small RNA fraction (<200 nt) isolated by size selection. For mRNA discovery: polyA+ RNA enrichment. QC Checkpoint: enrichment efficiency verified by Bioanalyzer trace.
3. Chemical Reduction — KBH₄ treatment under neutral conditions (3 hours). QC Checkpoint: reduction control sample included to confirm conversion efficiency. Input controls — input1 (no reduction, standard RT) and input2 (no reduction, skewed dGTP) — carried alongside for background assessment.
4. Library Construction and Sequencing — Adapter ligation with random molecular tag; HIV RT under optimized 1 mM dGTP / 100 μM dNTP; PCR amplification; sequencing on Illumina platform (NovaSeq 6000 or equivalent). QC Checkpoint: library yield, size distribution, and Q30 score ≥ 85%.
5. Data Processing and D Calling — Adapter trimming and molecular tag deduplication; alignment to tRNA reference database (GtRNAdb / mito-tRNA) and transcriptome; T→C mutation rate extraction per position; application of motif-specific calibration curves for D stoichiometry calculation. QC Checkpoint: positive control D sites in known tRNA positions verified; negative control (unmodified U sites) background rate assessed.
Sample
Sample Requirements and Shipping Instructions
Requirement
Details
Cell number
≥ 5 × 10⁶ cells (recommended for total RNA extraction)
Note: Sample quality is the single most important determinant of CRACI-Seq data quality. RNA degradation or genomic DNA contamination will reduce D detection sensitivity. Samples failing RNA integrity thresholds will be flagged and the client contacted before proceeding.
Deduplication using random molecular tag in 3′ adapter
tRNA alignment
Alignment to cytoplasmic and mitochondrial tRNA reference databases
D site identification
Per-position T→C mutation rate extraction; D stoichiometry calculation via motif-specific calibration
D annotation
Genomic position, tRNA gene and isotype, D-loop position (16/17/20/20a/20b/47), DUS enzyme assignment
D modification profile
Pie/donut chart of D distribution across tRNA species; heatmap of D stoichiometry across tRNAs × positions
Data delivery
Raw FASTQ, deduplicated BAM, D site table, annotation file, QC report, figures
Optional Advanced Analysis
Differential D analysis — Comparison of D stoichiometry between two or more experimental conditions (e.g., DUS knockdown vs control, treatment vs vehicle, disease vs normal), with statistical testing per D site
DUS writer assignment — Quantification of D stoichiometry changes upon individual DUS enzyme perturbation (siRNA or KO), enabling assignment of writer enzymes to specific D positions
Cross-species D conservation analysis — Comparison of D profiles across human, mouse, and/or plant samples to identify conserved and species-specific D sites
Mitochondrial D analysis — Focused D mapping in mitochondrial tRNAs, including mt-tRNAAsn, mt-tRNAGln, and mt-tRNALeu(UUR)
Demo
Representative Data: CRACI-Seq Demo Results
The data panels below represent the types of results delivered in a standard CRACI-Seq experiment.
Panel A — D Modification Profile (Pie/Donut Chart). Distribution of identified D sites across tRNA isotypes. Each segment represents one tRNA species, with segment area proportional to the number of D sites detected in that tRNA. This overview reveals which tRNAs carry the highest D modification load.
Panel B — D Stoichiometry Heatmap. Quantitative matrix showing D modification fraction (0–100%) for each tRNA (rows) at each D-loop position (columns). Positions 16 and 17 typically exceed 70% modification, position 20 > 60%, and position 20a ranges from 20–100% depending on the tRNA isotype.
Panel C — Per-Site D Stoichiometry Bar Chart. Comparison of D modification levels at individual positions between two experimental conditions (e.g., DUS KD vs control). Each D position is shown as a grouped bar pair, with statistical significance indicated. This analysis enables functional assignment of DUS enzymes to specific D positions.
Panel D — Mitochondrial tRNA D Map. Genomic map of D sites identified in mitochondrial tRNAs. Positions D16, D17, and D20 in mt-tRNAAsn, mt-tRNAGln, and mt-tRNALeu(UUR) are detectable with quantitative stoichiometry.
Panel E — Calibration Curve for D Quantification. Linear relationship between measured T→C mutation rate and expected D fraction across the NNDNN motif space. The calibrated quantification distinguishes CRACI from all prior D detection methods.
Panel F — T→C Misincorporation Trace at Representative D Site. Per-nucleotide mutation rate profile across a tRNA D-loop region, showing >90% T→C mutation at the D position with <5% background at flanking undmodified U positions.
Applications
Applications: Dihydrouridine in Health and Disease
Dihydrouridine modification is emerging as a functionally important epitranscriptomic mark with implications across multiple research areas.
DUS Enzymes in Cancer
DUS2L overexpression in non-small cell lung cancer suppresses ferroptosis through tRNACys hypomodification, reducing cysteine-rich metallothionein translation and glutathione synthesis. DUS1L expression levels correlate with glioblastoma patient survival and regulate translation through tRNATyr(GUA) processing. DUS4L has been shown tumorigenic in lung cancer models. CRACI-Seq provides the quantitative D mapping needed to characterize DUS enzyme activity in tumor samples and evaluate DUS inhibitors under development.
tRNA Biology and Translation Control
D modification is essential for proper D-loop folding and tRNA tertiary structure. Loss of D at specific positions alters tRNA stability, aminoacylation efficiency, and ribosome decoding rates. CRACI-Seq enables researchers to systematically map D under different growth conditions, stress responses, and DUS perturbation states to understand how D modulation affects the translational landscape.
Mitochondrial tRNA Modification
The discovery of D sites in human mitochondrial tRNAs opens a new area of mitochondrial epitranscriptomics. Mitochondrial tRNA modifications are linked to mitochondrial diseases, cardiomyopathies, and neurodegenerative disorders. CRACI-Seq is currently the only sequencing-based method capable of detecting and quantifying D in mitochondrial tRNAs.
mRNA Epitranscriptomics
D is now recognized as a rare but detectable modification in mRNA, opening questions about its function in post-transcriptional gene regulation — whether it affects RNA structure, stability, or translation efficiency. CRACI-Seq applied to polyA+ RNA represents the current best available tool for mRNA D discovery.
Cross-Species Comparative Epigenomics
CRACI has been validated in human, mouse, and Arabidopsis samples, revealing highly conserved D positions (D16/D17/D20/D47) across mammals and plants, with others (D20a) showing more species-specific patterns. This suggests fundamental roles for certain D modifications that may be relevant across model organisms.
Case
Case Study: Quantitative CRACI Defines the Human Dihydrouridine Landscape
Background: Dihydrouridine is among the most abundant RNA modifications in tRNA, yet the inability to quantify D at base resolution has limited our understanding of its distribution, writers, and regulatory logic. Prior methods relying on RT truncation could not determine D stoichiometry or distinguish closely spaced D sites in the tRNA D-loop.
Methods: Ju, He, et al. (University of Chicago / HKUST) developed CRACI — Chemical Reduction Assisted Cytosine Incorporation sequencing — using KBH₄ reduction of D to tetrahydrouridine followed by HIV reverse transcriptase with elevated dGTP/dNTP ratios (1 mM/100 μM). The method was applied to total RNA from HepG2 cells (human), mESCs (mouse), and Arabidopsis seedlings (plant), with validation by LC-MS/MS and DUS enzyme siRNA knockdown experiments across all four human DUS enzymes (DUS1L, DUS2L, DUS3L, DUS4L).
Results: CRACI achieved an average ~96% T→C conversion rate at D sites across all 256 NNDNN sequence motifs, with a background of <15% at unmodified U. The method identified and quantified D at six positions across 46 human cytoplasmic tRNAs (D16, D17, D20, D20a, D20b, D47). D16 and D17 consistently exceeded 70% modification, D20 and D47 exceeded 60%, while D20a ranged from 20–100% depending on tRNA isotype. Systematic DUS knockdown assigned each writer: DUS1L → D16/D17, DUS2L → D20, DUS3L → D47, DUS4L → D20a. DUS2L was further identified as the writer for all mitochondrial D sites (mt-tRNAAsn, mt-tRNAGln, mt-tRNALeu(UUR)). Quantitative analysis revealed that D20a negatively regulates installation of adjacent D20 — a cis-regulatory relationship invisible to truncation-based methods. D was confirmed present in mRNA but at very low abundance and stoichiometry (10–40%). Cross-species comparison showed D16/D17/D20/D47 are evolutionarily conserved, while D20a is mammal-specific.
Conclusion: CRACI is the first method to enable quantitative, base-resolution, transcriptome-wide dihydrouridine mapping. It resolves D sites inaccessible to prior techniques, assigns DUS writers with stoichiometric precision, and establishes a framework for studying D modification dynamics in development, stress, and disease.
Figure 2 from Ju et al., Nature Communications, 2025 — CRACI-Seq characterizes D sites across human cytoplasmic and mitochondrial tRNAs with quantitative stoichiometry.
Comparison
Comparison: CRACI-Seq vs Other D Detection Methods
Feature
CRACI-Seq
D-seq
AlkAniline-Seq
Rho-seq
Detection signal
T→C misincorporation
RT truncation (stop)
Aniline cleavage + stop
RT truncation
Base resolution
Yes
Partial
Yes
Partial
Quantitative stoichiometry
Yes (calibration-based)
No
No
No
Reads through clustered D sites
Yes (internal mutation)
No
No
No
D detection in mRNA
Yes (low abundance)
Yes
Limited
Limited
Background control
Input1 + input2 dual control
Limited
Limited
Limited
Motif bias
Low (calibrated across 256 motifs)
Unknown
Sequence-dependent
Unknown
Commercial service available
Yes (CD Genomics)
No
No
No
Which Method to Choose: CRACI-Seq is the appropriate choice when quantitative D stoichiometry is required — whether for comparing D levels between conditions, assigning DUS writers, or detecting D in low-abundance contexts such as mRNA. If only binary D presence/absence is needed at well-characterized tRNA positions, truncation-based methods may be sufficient. However, for any study requiring modification dynamics, stoichiometric comparison, or confident detection in densely modified D-loop regions, CRACI-Seq provides information that older methods cannot deliver.
1. Ju CW, Li H, He C, et al. Quantitative CRACI reveals transcriptome-wide distribution of RNA dihydrouridine at base resolution. Nature Communications. 2025;16:8863. doi:10.1038/s41467-025-63918-w.
2. Matsuura T, et al. Human DUS1L catalyzes dihydrouridine modification at tRNA positions 16/17, and DUS1L overexpression perturbs translation. Communications Biology. 2024;7:1238. doi:10.1038/s42003-024-06942-8.
3. Finet O, Yague-Sanz C, Marchand V, Hermand D. The Dihydrouridine landscape from tRNA to mRNA: a perspective on synthesis, structural impact and function. RNA Biology. 2022;19(1):735–750. doi:10.1080/15476286.2022.2078094.
4. Draycott AS, Schaening-Burgos C, Rojas-Duran MF, et al. Transcriptome-wide mapping reveals a diverse dihydrouridine landscape including mRNA. PLOS Biology. 2022;20(5):e3001622. doi:10.1371/journal.pbio.3001622.
For research use only. Not for use in diagnostic procedures.
